Throughout this application various publications are referenced by Arab numerals within square parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
When producing recombinant proteins in eukaryotic cells for biopharmaceutical and biotechnological purposes, the level of expression is a central parameter which controls the economy of the project. When the desired protein is present in the culture medium or cellular extract at low concentrations, its recovery will entail more extensive protein purification and lower final yield. However, high constitutive expression can in some instances lead to counterselection of the expressing cells during their proliferation phase, obstructing expression of a pharmaceutically or technologically relevant secreted protein to high levels in cell culture. Hence, there is a critical need for efficient and regulated expression vectors.
Improving the design of the expression vectors is also critical for gene therapy as illustrated by the often suboptimal levels of expression which have been observed in clinical trials. In this context, transgenic animals provide an appropriate model for testing gene therapy constructs, where an ability to regulate expression is of paramount importance.
In the long term, transgenic animals or plants will provide an alternative to large scale cell culture for the production of massive amounts of proteins or other gene products such as RNA and this production will require careful regulation of expression of the gene encoding a desired product. With the development of gene transfer techniques that allow the generation of transgenic animals or plants came the possibility of producing transgenic livestock functioning as animal bioreactors as an alternative strategy to cell culture systems for protein production [1]. For instance, protein secretion in the milk of large mammals could provide a cost-effective route for the production of large amounts of valuable proteins. As yet this technology is still in development and needs optimization, and there is a general requirement for methods to improve productivity.
Regulated expression could be achieved by several routes. Efforts to improve the design of expression vectors for biotechnological purposes have focused on transcriptional control [1–4] in an attempt to achieve both high levels of transcription and externally regulatable transcription, and to some extent by control of translation. Large-scale cell culture production of biopharmaceutical generally is accomplished by use of constitutive high-level expression vectors [5, 6]. For expression of heterologous proteins that are toxic, or impose a negative effect on cell growth, a regulated expression system is highly desirable [5, 6]. This will allow for propagation of the production cells to high density before turning on expression of the desired protein. This was done previously by use of vectors with inducible transcription [7–11]. In most of these systems, however, the range over which expression can be controlled remains limited, thus inviting improvement.
This invention deals with another major level of control of gene expression which has received little attention up to now: mRNA splicing, which is the processing of precursor transcripts into mature mRNA containing only exon sequences, by excision of introns at the RNA level in the cell nucleus. The mRNA splicing step is a good candidate for control since evidence exists that this mechanism functions in vivo [12, 13]. There are several examples of genes requiring a splicing event for mRNA production [24] and an intron generally is included in pharmaceutically employed expression vectors [5, 6]. For complementary DNA (cDNA) expression, the contribution of the intron to final product formation seems to be cDNA-specific but the mechanism of intron action remains largely unknown [25]. To date, little effort has been directed at regulation expression of genes for biotechnological use or gene therapy at the mRNA splicing step. Regulation of mRNA splicing would be useful for regulating expression of genes that have been transferred, be it into cell lines, the germline or somatic tissues.
Expression of several cytokine genes is highly regulated at splicing of precursor transcripts [12, 13, 27–29]. Thus, shortly after the onset of induction of human interleukin-2 (IL-2) and interleukin-1β (IL-1β) genes, the flow of nuclear precursor transcripts into mature mRNA becomes blocked despite the fact that transcription, once activated by an inducer, continues unabated for an extensive period. Expression of IL-2 and IL-1β mRNA is superinduced by two orders of magnitude in the presence of translation inhibitors, without a significant increase in primary transcription or mRNA stability. Instead, splicing of precursor transcripts is greatly facilitated [13, 27].
Expression of the human tumor necrosis factor-α (TNF-α) gene is also regulated at splicing [13]. 2-Aminopurine (2-AP) blocks expression of TNF-α mRNA in primary human lymphoid cells. An adenine isomer, 2-AP inhibits specific kinases that phosphorylate the α-subunit of eukaryotic translation initiation factor 2 (eIF2α) [17], including the RNA-activated protein kinase, PKR [30]. 2-AP does not inhibit human TNF-α gene expression at transcription, nor does it affect mRNA stability. Instead, splicing of short-lived TNF-α precursor transcripts into mRNA is blocked when 2-AP is present during induction, causing pre-mRNA to accumulate at the expense of mRNA; stability of TNF-α precursor transcripts is unaffected [13]. 2-AP blocks splicing of TNF-α precursor transcripts at multiple splice junctions. Neither the human IL-1β nor TNF-β gene shows such regulation. A 2-AP-sensitive component, expressed in functional form before induction, regulates splicing of TNF-α mRNA [13].
PKR, an RNA-activated Ser/Thr protein kinase, is a major negative regulator of translation [14, 15]. PKR is expressed constitutively in most cells but is induced by viruses, double-stranded RNA (dsRNA) and interferons [16]. Activation of PKR requires its trans-autophosphorylation which is facilitated by RNA, especially by dsRNA [15]. PKR phosphorylates eIF2α, blocking GDP/GTP exchange [17] and preventing the recycling of eIF2 between rounds of initiation of translation [18]. Thus, activation of PKR triggers an inhibition of protein synthesis. Dominant-negative mutants of PKR have been described that inhibit trans-autophosphorylation of the wild type enzyme, obligatory for its activation [19–21].
Activation of PKR requires its dimerization on RNA [22, 23] and thereby depends critically on its binding to RNA [23]. PKR contains two tandem double-stranded RNA binding motifs found in diverse proteins such as Drosophila staufen, ribosomal protein S5 and E. coli RNase III [26]. Perfectly matched dsRNA having the A conformation as well as certain other RNAs, including hepatitis delta agent RNA [31], reovirus S1 3′-UTR [32] and human α-tropomyosin 3′-UTR [33] activate PKR in vitro while adenovirus VA RNA [34] and Alu RNA bind to PKR and thereby inhibit its activation [35]. Both the activation of PKR and its inhibition require highly ordered RNA structures, rather than a specific sequence. Certain highly structured RNAs can be activators or inhibitors of PKR even when they contain imperfectly matched base-paired domains, such as human delta hepatitis agent RNA [26] or VA RNA [34]. The RNA-binding domain in PKR [36] requires 11–13 bp of dsRNA for binding [22, 37, 38] and can tolerate non-Watson-Crick structures [39]. Moreover, noncontiguous short helices of RNA can cooperate in binding of PKR and thereby, in its activation [39].
PKR was detected in cell nucleoplasm in an underphosphorylated state [40]. Upon induction by interferon, aggregates of PKR are colocalized with interchromatin granule clusters [41] known to contain significant amounts of spliceosomal components and to be involved in spliceosome assembly, sorting and recycling [42]. Modified splicing factors are recruited from these clusters into perichromatin fibrils where gene transcription occurs, facilitating cotransciptional RNA processing [42]. However, no functional connection between PKR and splicing was reported prior to this invention.
The TNF-α3′-untranslated region (3′-UTR) has multiple roles in regulating expression of TNF-α mRNA. It downregulates the murine TNF-α promoter at transcription [43] and harbors an AU-rich determinant of mRNA instability [44]. This AU-rich motif mediates translational inhibition by IL4 and IL-13 [45] and activation of translation by lipopolysaccharide [14] which induces formation of protein complexes that bind specifically to the nonanucleotide UUAUUUAUU [46]. However, no functional role for the TNF-α3′-UTR, or for a 3′-UTR as such, in the regulation of mRNA splicing was reported prior to this invention.
This invention describes the introduction of a novel cis-acting element, preferably within an expression construct, into a gene of interest, in order to render splicing of mRNA expressed by this gene dependent on the activation of an RNA-activated eIF2α kinase, thereby imparting on the expression of this gene a regulation at the mRNA splicing step by the novel cis-acting sequence element, through manipulation of the expression vector on one hand and application of methods known in the art to modulate the expression and/or activity of the RNA-activated eIF2α kinase in the recipient cells or organism on the other hand. Thus, the invention provides a novel solution to attain a regulated system for production of proteins of interest and to optimize expression and yield of such protein.